Morphologically differentiated sex chromosomes in neotropical freshwater fish

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1 Genetica 111: , Kluwer Academic Publishers. Printed in the Netherlands. 91 Morphologically differentiated sex chromosomes in neotropical freshwater fish L.F. de Almeida Toledo 1 &F.Foresti 2 1 Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, Caixa Postal , São Paulo, SP, Brasil ( lftoledo@usp.br); 2 Departamento de Morfologia, Instituto de Biociências, UNESP, Campus de Botucatu, , Botucatu, Brasil Key words: fish cytogenetics, neotropical fishes, sex-chromosomes, XX-XY, ZZ-ZW Abstract A general survey of the occurrence of morphologically differentiated sex chromosomes in the neotropical freshwater fishes is presented. The total number of 32 occurrences involving simple XX-XY and ZZ-ZW, and multiple X 1 X 2 Y, XY 1 Y 2 and ZW 1 W 2 sex chromosome systems is described, with comments on the aspects of sex chromosome evolution in this fish fauna. The occurrence of different sex chromosome systems in related species of the same genus, or in different populations of the same nominal species, involving male and sometimes female heterogamety, and differences in the molecular composition of sex-linked heterochromatin, are considered as indicative of the early stage of sex chromosomes evolution in fish. Introduction The neotropical freshwater fish fauna is the most morphologically diversified among the epicontinental fish faunas in the world (Vari & Malabarba, 1998) and its number of species may exceed 8,000 (Schaefer, 1998). About 10% of these species have been analyzed cytogenetically, that is approximately 900 species, belonging to 252 genera and 44 families (Oliveira, 2000). Paralleling their extraordinary number and morphological diversity, neotropical freshwater fish species present considerable inter and intraspecific chromosome variability (Oliveira et al., 1988) with diploid numbers that vary from 2n = 20 found in Pterolebias longipinnis, a Cyprinodontiformes species (Post, 1965), to 2n = 132 in Corydoras aeneus, a Siluriformes species (Scheel, Simonsen & Gyldenholm, 1972). Considering the sex chromosomes, fish are characterized by a remarkable variability of sex determination systems. While mammals are characterized by XX-XY and birds by ZZ-ZW sex chromosomes, eight types of sex chromosome systems have already been described in fish (Tave, 1993), involving both morphologically undifferentiated and differentiated homologues, in simple or multiple systems with male or female heterogamety. The first evidence of morphologically differentiated sex chromosomes in neotropical freshwater fish was the discovery of clearly visible neo-y chromosomes in males of two unnamed Mexican fish species (Uyeno & Miller, 1971, 1972). In the following years, with the growing development of cytogenetic techniques, new occurrences of differentiated sex chromosomes have been described. Considering our present revision, differentiated sex chromosomes may be considered to occur in a total number of 32 species, that is, in about 4.0% of the total number of species already analyzed cytogenetically. A very interesting feature of the sex chromosomes in the neotropical freshwater fish is that, in spite of the relatively low number of occurrences, a wide range of sex chromosome systems is present, involving simple XX-XY and ZZ-ZW, and multiple X 1 X 2 Y, XY 1 Y 2 and ZW 1 W 2 systems. In most cases, the difference between the sex homologues is due to heterochromatin addition/deletion to the X, the Y, the Z or the W chromosome. The morphological differentiation of these chromosomes is very well studied in ZZ-ZW systems in which the W chromosome has a heterochromatic region not present

2 92 in the Z chromosome (Haaf & Schmid, 1984; Galetti & Foresti, 1986; Feldberg et al., 1987; Bertollo & Cavallaro, 1992; Moreira-Filho et al., 1993). Multiple systems, on the other hand, have originated by Robertsonian or tandem fusion, sometimes resulting in heterochromatin loss (Almeida-Toledo et al., 2000a) or by chromosome fission. Differentiated sex chromosomes present a very peculiar distribution among fish species. The same system may be found in all the species of a group, as is the case of the ZZ-ZW system which is found in all the species in genus Triportheus (Bertollo & Cavallaro, 1992; Artoni, 1999); or it may be found in part of a larger group, as in six out of more than 20 cytogenetically analyzed and probably monophyletic, ZZ-ZW species from the genus Leporinus (Galetti & Foresti, 1986). Most of the occurrences, however, are sporadic, as in Semaprochilodus, a Characidae genus for which sex chromosomes are present only in one species of the group, S. taeniurus (Feldberg et al., 1987). The most interesting feature of the sex chromosome distribution in fish groups is that in these sporadic occurrences, different systems for example male and female heterogamety and simple or multiple systems, may be found in closely related species of the same genus, or in different populations of the same species. Such is the case in Eigenmannia (L.F. Almeida-Toledo & F. Foresti, submitted) and in Hoplias (Bertollo et al., 2000) and probably characterizes early stages and different strategies of incipient sex chromosome differentiation. XX-XY systems A reference list of the occurrence of morphologically differentiated sex chromosomes in gonochoristic teleosteans was provided by Chourrout (1988) and among sixty species reported, 25 had XX-XY sex chromosomes. These first reports include the welldocumented occurrences of XX-XY in a deep-sea species (Chen & Ebeling, 1966) and in a Salmonidae species (Thorgaard, 1977). However, some of the reported occurrences, mainly for neotropical freshwater species, were put in doubt (Moreira-Filho, Bertollo & Galetti-Jr, 1993) or discarded (Mestriner, Bertollo & Galetti, 1995) by further studies. On the other hand, some new occurrences of the XX-XY sex chromosome system have been detected with the use of banding techniques, as for example in the lake trout Salvelinus namaycush (Phillips & Ihssen, 1985). Table 1. XX-XY sex chromosomes in neotropical freshwater fish Species 2n Sex Reference chromosome system STERNOPYGIDAE Eigenmannia virescens 38 XX-XY 1,5 HYPOPTOPOMATINAE Pseudotocinclus tietensis 54 XX-XY 2 POECILIIDAE Poecilia reticulata 46 XX-XY 3 ERYTHRINIDAE Hoplias malabaricus 42 XX-XY 4 References: 1. Almeida-Toledo, Foresti and Toledo-Filho, 1988; Andreata et al., 1992; 3. Nanda et al., 1990; 4. Born and Bertollo, 2000; 5. Almeida-Toledo et al., submitted. Other occurrences have been found by meiotic analysis, such as in the rainbow trout Oncorhyncus mykiss (Oliveira et al., 1995) and in the tilapia, Oreochromis niloticus, as suggested by Foresti et al. (1993) and confirmed by Carrasco et al. (1999). Among the neotropical freshwater species we now list four species for which clearly documented occurrences of XX-XY sex chromosomes were reported (Table 1): Eigenmannia virescens (Almeida-Toledo, Foresti & Toledo-Filho, 1988), Poecilia reticulata (Nanda et al., 1990), Pseudotocinclus tietensis (Andreata et al., 1992) and Hoplias malabaricus (Born & Bertollo, 2000). The interesting feature in these cases is that the differentiation of the homologues was attained either by Y or X heterochromatinization: in both E. virescens and in H. malabaricus the heterochromatinization occurred in the X chromosome. For P. reticulata and for P. tietensis, addition of a heterochromatic block occurred in the Y chromosome. In Eigenmannia, an early stage of differentiation of an XX-XY system with heterochromatin addition to the X chromosome was detected, involving two geographically related populations of Eigenmannia virescens (Gymnotiformes, Sternopygidae) from which the two otherwise identical cytotypes differ by the presence or lack of well-differentiated sex chromosomes (L.F. Almeida-Toledo et al., submitted). In this case, in one of the cytotypes of E. virescens (cytotype A), the acrocentric X chromosome presents an additional block of heterochromatin in its terminal portion without a corresponding region in the acrocentric Y chromosome, while the other cytotype (cytotype B) has no differentiated sex homologues (Figure 1). The additional heterochromatic segments in the sex homo-

3 93 Figure 1. C-banded metaphases of Eigenmannia virescens. (A) cytotype A female with large heterochromatic blocks in the X-chromosomes (B) cytotype A female with polymorphic heterochromatic blocks in the X-chromosomes; (C) cytotype A male; (D) cytotype B with undifferentiated sex chromosomes. The arrows indicate X-chromosomes (Almeida-Toledo et al., submitted). logues seem to result from DNA amplification. After BrdU incorporation, it was possible to identify the same banding pattern in the euchromatic region of X and Y-chromosomes in cytotype A andalsointhe corresponding chromosome pair in cytotype B. There is no indication of any alteration in the replication pattern of the euchromatic region of the sex chromosomes in the two populations. A polymorphic size variation of the heterochromatic segment present in the sex chromosomes was detected in cytotype A, but in all cases males always presented a reduction of heterochromatin in relation to females (Figure 1(B)). Hoplias malabaricus, like E. virescens, presentsa heterochromatic block in the submetacentric X chromosome (Born & Bertollo, 2000). In this case also, the occurrence may be considered to be in an early stage of diversification, since related populations of the same species do not display differentiated sex chromosomes or present other sex chromosome systems, as will be discussed later.

4 94 In Poecilia reticulata (Poeciliidae, Cyprinodontiformes) a heterochromatic region with differential accumulation of (GACA) 4 repeats was detected in specific Y chromosomes of a laboratory strain, indicating the occurrence of an XX-XY mechanism of sex determination (Nanda et al., 1990). According to the authors, the results obtained for these lineages may point to early stages in morphological differentiation of vertebrate sex chromosomes in general. In Pseudotocinclus tietensis (Siluriformes, Loricariidae), an XY heteromorphic sex chromosome pair was detected, and the metacentric Y chromosome presented a conspicuous heterochromatic region in the short arm (Andreata et al., 1992). It is important to notice that from the initial descriptions of XX-XY systems, for all the confirmed occurrences, the differentiation of the heteromorphic pair was attained by processes of addition/deletion of heterochromatin. Reported occurrences of differentiated XY chromosomes for which the homologue differentiation was attributed to pericentric inversion could not be entirely proved and, in some cases, were reconsidered when detailed meiotic chromosome analysis was performed. This was, for instance, the case of Leporinus lacustris (Mestriner et al., 1995). In E. virescens, the X-linked heterochromatic segment is brightly stained with chromomycin A 3 and mithramycin. It is also late replicating in BrdU treated chromosomes, is digested in situ by the restriction enzyme AluI, and resistant to HindIII digestion (L.F. Almeida-Toledo et al., submitted). In Hoplias malabaricus on the other hand, this segment is DAPI positive (Born & Bertollo, 2000). ZZ-ZW systems The ZZ-ZW simple system with female heterogamety is the most frequent among neotropical freshwater fish, with 20 occurrences already reported (Table 2). The ZZ-ZW system differs from the previously discussed XX-XY system in being well established in certain fish genera such as Leporinus and Triportheus. This latter genus presents female heterogamety in all the species already analyzed, a situation that is still unique in the neotropical fish fauna, that is, an entire group presenting differentiated sex chromosomes. In these two cases, the presence of such a distinctive characteristic seems to imply a close phylogenetic relationship among the species involved. In the genus Leporinus (Characiformes, Anostomidae) containing fish with a wide occurrence in the neotropical region, sex chromosomes were detected in six of the 20 species already analyzed cytogenetically. The sex chromosome differentiation in these six species is due to the presence of a ZW heteromorphic pair in females, in which one small subtelocentric chromosome is the Z and one large subtelocentric chromosome with a large C-band positive block that occupies nearly the entire length of the long arm is the W (Galetti & Foresti, 1986, 1987; Galetti, Lima & Venere, 1995) (Figure 2). The geographic distribution of the ZW species of Leporinus including species from the upper Paraná river system and the São Francisco river system seems to indicate that this sex chromosome system evolved only once in this group, from a very ancient ancestor, and before the separation of these two river systems in South America (Galetti, Lima & Venere, 1995). This monophyletic origin is corroborated by the homologies exhibited concerning relative size, centromere position and amount and distribution of heterochromatin of the W chromosome in Leporinus species (Koehler et al., 1997). In the genus Triportheus (Characiformes, Characidae), on the other hand, a ZZ-ZW system is present in all of the eight species and populations that have already been analyzed cytogenetically. A well differentiated ZW pair is present in this group, the W chromosome being a small almost entirely heterochromatic metacentric and the Z a large metacentric (Bertollo & Cavallaro, 1992; Artoni, 1999). Morphological differentiation of the W chromosome has also been observed among populations of one nominal species of this genus in samples from different river basins (Artoni, 1999), indicating their probable origin from a common ancestor. Leporinus and Triportheus are the only two genera for which a female sex chromosome heteromorphism is a fixed characteristic within a group of species or all of the species in a genus. Other ccurrences of the ZW system in neotropical freshwater fishes are sporadic (Table 2). It has been observed in Parodon hilarii (Characiformes, Parodontidae) (Moreira-Filho, Bertollo & Galetti, 1993), Semaprochilodus taeniurus (Characiformes, Prochilodontidae) (Feldberg et al., 1987), Characidium fasciatum (Characiformes, Characidae) (Maistro et al., 1998), Microlepidogaster leucofrenatus (Siluriformes, Loricariidae) (Andreata et al., 1993), and Poecilia sphenops var. melanistica (Cyprinodontiformes, Poeciliidae) (Haaf & Schmid, 1984). In all these cases, the W differs from the Z by the presence of conspicuous heterochromatin blocks. The only exception to this pattern is found in

5 95 Table 2. ZZ-ZW sex chromosomes in neotropical freshwater fish Species 2n Sex chromosome Reference system ANOSTOMIDAE Leporinus elongatus 54 ZZ-ZW 1 Leporinus obtusidens 54 ZZ-ZW 1 Leporinus reinhardti 54 ZZ-ZW 2 Leporinus macrocephalus 54 ZZ-ZW 2 Leporinus trifasciatus 54 ZZ-ZW 3 Leporinus conirostris 54 ZZ-ZW 3 Leporinus cf. elongatus 54 ZZ-ZW 4 CHARACIDAE Triportheus albus 52 ZZ-ZW 5 Triportheus signatus 52 ZZ-ZW 5 Triportheus elongatus 52 ZZ-ZW 5 Triportheus cf. elongatus 52 ZZ-ZW 7 Triportheus flavus 52 ZZ-ZW 5 Triportheus guentheri 52 ZZ-ZW 6 Triportheus paranense (MT) 52 ZZ-ZW 7 Triportheus paranense (MS) 52 ZZ-ZW 7 Characidium fasciatum 50 ZZ-ZW 8 PARODONTIDAE Parodon hilarii 54 ZZ-ZW 9 PROCHILODONTIDAE Semaprochilodus taeniurus 54 ZZ-ZW 10 LORICARIIDAE Hypostomus sp. 64 ZZ-ZW 11 Microlepidogaster leucofrenatus 54 ZZ-ZW 12 POECILIIDAE Poecilia sphenops var. melanistica 46 ZZ-ZW 13 References: 1. Galetti et al.,1981; 2. Galetti and Foresti, 1986; 3. Galetti, Lima and Venere, 1995; 4. Molina, Schmid and Galetti, 1998; 5. Falcão, 1988; 6. Bertollo and Cavallaro, 1992; 7. Artoni, 1999; 8. Maistro et al., 1998; 9. Moreira-Filho, Bertollo and Galetti, 1993; 10. Feldberg et al., 1987; 11. Artoni et al., 1998; 12. Andreata et al., 1993; 13. Haaf and Schmid, Hypostomus sp. (Siluriformes, Loricariidae) (Artoni et al., 1998). In this case a ZZ-ZW system is present, but a heterochromatin block occurs in the interstitial region of the acrocentric Z chromosomes, the W being a small metacentric with no detectable heterochromatin differentiation. The W-linked heterochromatic region is DAPI positive in Poecilia sphenops var. melanistica (Haaf & Schmid, 1984), but either G + CrichorA+ Trich regions have been observed in sex chromosomes of Leporinus (Molina, 1995; Koehler et al., 1997). Multiple sex chromosome systems Multiple sex chromosome systems usually arise as a result of rearrangements involving sex chromosomes and autosomes, either by centric fusion, reciprocal translocation between metacentric chromosomes, or centric fission; tandem fusions may also be involved in this process (White, 1973). Multiple sex chromosome systems have been detected in seven species of freshwater fish from the neotropical region (Table 3), with male X 1 X 1 X 2 X 2 - X 1 X 2 Y and XX-XY 1 Y 2, or female ZZ-ZW 1 W 2 heterogamety. In five species, X 1 X 1 X 2 X 2 -X 1 X 2 Y systems were reported: initially, in one Mexican species of Cyprinodontidae where the Y chromosome originated through a Robertsonian fusion with some chromatin addition (Uyeno & Miller, 1971) and in one species of Goodeidae in which the Y chromosome also originated by fusion of the sex chromosome and one autosome (Uyeno & Miller, 1972), with no detectable chromatin addition. Similar occurrences were reported

6 96 Figure 2. C-banded metaphases of Leporinus macrocephalus. (A) female; (B) male. The arrow indicates the W-chromosome. Table 3. Multiple systems of sex chromosome differentiation in neotropical freshwater fish Species 2n Sex chromosome Reference system PARODONTIDAE Apareiodon affinis F-55 F-ZW 1 W 2 1,2 M-54 M-ZZ ERYTHRINIDAE Hoplias malabaricus F-40 F-X 1 X 1 X 2 X 2 3 M-41 M-X 1 X 2 Y Hoplias sp. F-40 F-XX 4 M-41 M-X 1 X 2 Y STERNOPYGIDAE Eigenmannia sp.2 F - 32 F - X 1 X 1 X 2 X 2 5 M-31 M-X 1 X 2 Y HYPOPOMIDAE Brachyhypopomus F-42 F-X 1 X 1 X 2 X 2 6 pinnicaudatus M-41 M-X 1 X 2 Y CYPRINODONTIDAE unnamed Mexican Sp. F - 48 F - X 1 X 1 X 2 X 2 7 M-47 M-X 1 X 2 Y GOODEIDAE unnamed Mexican sp F - X 1 X 1 X 2 X 2 8 M-X 1 X 2 Y F = female; M = male. References: 1. Moreira-Filho, Bertollo and Galetti, 1980; 2. Moreira-Filho, Bertollo and Galetti, 1993; 3. Bertollo, Takahashi and Moreira-Filho, 1983; 4. Dergan and Bertollo, 1990; 5. Almeida-Toledo, Foresti and Toledo-Filho, 1984; 6. Almeida-Toledo et al., 2000b; 7. Uyeno and Miller, 1971; 8. Uyeno and Miller, 1972.

7 97 Figure 3. Chromosome plates of Eigenmannia sp.2. (A) Giemsa stained metaphase of a female; (B) Giemsa-stained metaphase of a male; (C) Mithramycin-stained male metaphase. The arrows indicate the Y chromosome (Almeida-Toledo et al., 2000a). in Eigenmannia sp.2 (Gymnotiformes, Sternopygidae) (Almeida-Toledo, Foresti & Toledo-Filho, 1984, Almeida-Toledo et al. 2000a) and in Hoplias sp. (Erythrinidae, Characiformes) (Bertollo, Takahashi & Moreira, 1983). In Eigenmannia, as been observed in the Mexican species, a Robertsonian translocation was the possible origin of the large neo-y chromosome; in Hoplias, a complex rearrangement, possibly involving a pericentric inversion and a chromosome translocation seems to have occurred (Bertollo et al. 1997). A second occurrence in Gymnotiformes, and the fourth in neotropical freshwater fish of X 1 X 1 X 2 X 2 - X 1 X 2 Y sex chromosome systems involving Robertsonian translocation, was reported more recently in Brachyhypopomus pinnicaudatus (Almeida-Toledo et al., 2000b). Besides these occurrences of X 1 X 2 Ysystems,a ZZ-ZW 1 W 2 system and an XX-XY 1 Y 2 system were reported. The ZZ-ZW 1 W 2 system was detected in Apareiodon affinis (Characiformes, Parodontidae). In this species, males have one chromosome less than females, probably as result of a fission process involving the largest chromosome pair, since in a closely related species the corresponding pair is homomorphic in both males and females (Moreira-Filho et al., 1980). The only reported occurrence of an XX-XY 1 Y 2 system was detected in one cytotype of the Hoplias malabaricus (Characiformes, Erythrinidae) complex (Bertollo, Takahashi & Moreira-Filho, 1983; Bertollo et al., 2000) in which males have one chromosome more than females, probably originating through a fusion/fission process. Heterochromatin loss in the neo-y formation, probably related to the occurrence of a chromosome translocation process, was observed in Eigenmannia sp.2 and the missing region was G + C rich (Figure 3) (Almeida-Toledo et al., 2000a). Conversely, in Hoplias malabaricus, no chromosome loss apparently resulted from the tandem translocation involved in the neo-y chromosome formation, as shown by C- banding analysis (Bertollo et al., 1997). The same is true for Brachyhypopomus pinnicaudatus (Almeida- Toledo et al., 2000b). In all these cases, disturbance in the meiotic chromosome pairing process is caused by the chromosome rearrangement, as seen in H. malabaricus (Bertollo & Mestriner, 1998). General comments A general view of the morphologically differentiated sex chromosomes in neotropical freshwater fish indicates a relatively wide occurrence of the ZZ-ZW system as a more constant and fixed presence in two groups of Characiformes, that is, in part of family Anostomidae and in the entire subfamily Triportheinae. Sporadic occurrences in other fish families of Characiformes, (Prochilodontidae and Parodontidae) also occur, including a unique occurrence in the neotropical fish fauna, of a multiple ZW 1 W 2 system in a Parodontidae species. Male heterogamety was not detected in the species already described in the group including families Prochilodontidae, Parodontidae and Anostomidae. This points to a possible tendency for female heterogamety that could be a fixed characteristic in this group. According to Vari (1983), Prochilodontidae and Parodontidae are closely related to Anostomidae, and the presence of the same sex chromosome system in species of this group reinforces their relationship from the genetic point of view. This tendency is not

8 98 observed when all the Characiformes species are considered: in Erythrinidae, a family belonging to this order, only male heterogamety was described (Bertollo et al., 2000). Most of the reported occurrences of XX-XY systems were sporadic, but sometimes multiple sex chromosomes with male heterogamety were detected in populations or species related to those presenting XX-XY systems. This was the case of Sternopygidae (Almeida-Toledo, Foresti & Toledo-Filho, 1984, 1988) and Erythrinidae (Bertollo et al., 2000). For these two fish families, both simple XX-XY and multiple X 1 X 1 X 2 X-X 1 X 2 Y systems were described in closely related species. In Poeciliidae, a well studied group that presents a wide variety of sex determination mechanisms which span from simple ZZ-ZW or ZZ-ZW systems to polyfactorial sex determination (Volff & Schartl, this volume), only early stages of morphologically differentiated sex chromosomes have been detected (Haaf & Schmid, 1984; Nanda et al., 1990) and both male and female heterogamety were found. Experiments carried out by Nakayama et al. (1994) using sex-specific clones isolated from Leporinus elongatus and hybridized with DNA from 24 adults of the same species have revealed the presence of one female and two male individuals with opposite hybridization, that is, three specimens had atypical W-chromosomes. The results were probably due to a possible loss of the sex-related insert used as a probe (Nakayama et al., 1994). However these investigators presented an alternative suggestion that these atypical Ws could have been the product of a cross-over involving the sex-linked region, thereby providing evidence that might explain how male and female heterogamety has appeared several times in different fish subtaxa (Nakayama et al., 1994). As further support of this idea, sex-chromosome crossovers were also reported in Xiphophorus maculatus in the region of the X and Y chromosomes that encodes several important traits, including the determination of sex (Gutbrod & Schartl, 1999). Fluorochrome analysis of the heterochromatic regions of sex chromosomes was carried out in some species. In the family Anostomidae Z and W chromosomes of Leporinus obtusidens are characterized by a high GC content, while AT sequences have accumulated in the heterochromatic portions of Z and W chromosomes of L. elongatus (Koehler et al., 1997). A difference in the amount of heterochromatin associated with the sex chromosomes was detected between males and females in Eigenmannia sp.2: a CMA3 positive and G + C rich heterochromatic portion of one of the chromosomes involved in the Robertsonian fusion that gave origin to the bi-armed Y was lost, so that the female X 1 and X 2 chromosomes have more G + Crich heterochromatin than the corresponding neo-y in the male (Almeida-Toledo et al., 2000a). In another Eigenmannia species, E. virescens, the heterochromatic region associated with the X chromosome is G+C rich. The detection of sex-chromosomes linked A + Trich heterochromatin in the pericentromeric region of the neo-y chromosome of Eigenmannia sp.2 and B. pinnicaudatus (Almeida-Toledo et al., 2000a,b) indicates that these regions, probably present in the original translocated acrocentrics, were not lost during the fusion process. A + T rich heterochromatin associated with the sex chromosomes has already been reported to occur in Poecilia by Haaf and Schmid (1984) and in the Hoplias malabaricus X-chromosome (Born & Bertollo, 2000). The significance of the loss of G + C rich heterochromatin in Eigenmannia sp.2 males and the conservation of A + T heterochromatin associated with the sex chromosomes in these species remains to be explained. The finding that, in addition to the occurrence of different systems of sex chromosomes, differences also occur in the molecular nature of the heterochromatin associated with the sex-related regions, reinforces the idea that sex chromosomes may have evolved independently and in different ways in the various fish lineages, resulting in the rich variability of sex chromosome systems in the neotropical fish fauna. Transposable elements that are widespread in fish, one of them having been identified in the sex chromosomes of Xiphophorus (Volff et al., 1999) could also be involved in the high degree of variability of the sex chromosomes in this group. The occurrence of such variable forms of chromosome heteromorphism and of different mechanisms of sex determination in closely related species, most of them at an early stage of evolution, seems to be well tolerated in fish, although not the case with higher vertebrates. An indication of this labiality of sex determination is found in sex reversal experiments carried out in aquaculture programs (Mair et al., 1991; Rosenstein & Hulata, 1994 among others). In the neotropical species Leporinus macrocephalus, which presents morphologically well differentiated sex chromosomes, histologically analyzed male gonads were observed in genetically ZW females that had been

9 99 submitted to methyltestosterone treatment (E. Toriyama & F. Foresti, unpublished). The growing use of a molecular cytogenetic approach in the neotropical freshwater fishes, mainly involving replication banding, in situ hybridization of selected sequences, and other high resolution cytogenetic techniques, may help provide a better understanding of the chromosome evolutionary trends in fishes of the neotropical region, and may also allow a closer view of the molecular organization of fish sex chromosomes. Acknowledgements The authors are grateful to Maria de Fátima Z. Daniel- Silva for her helpful assistance and to Dr Ann Stocker for the critical reading of the manuscript. Funds supporting this study were provided by FAPESP and CNPq. Copyright permission was obtained for publication of Figure 3(C). References Almeida-Toledo, L.F., F. Foresti, E.V. Péguignot & M.F.Z. 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